Human SMN-like protein

ABSTRACT

The invention provides a human SMN-like protein (HSLP) and polynucleotides which identify and encode HSLP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for treating or preventing disorders associated with expression of HSLP.

[0001] This application is a divisional application of U.S. applicationSer. No. 09/571,078, filed May 15, 2000, now U.S. Pat. No. 6,620,783,issued on Sep. 16, 2003, which is a continuation in part (CIP)application of U.S. application Ser. No. 09/028,327 filed Feb. 24, 1998,now U.S. Pat. No. 6,130,064, issued Oct. 10, 2000, the contents all ofwhich are hereby incorporated by reference herein.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a human SMN-like protein and to the use of these sequences in thediagnosis, treatment, and prevention of neurological, reproductive, andcell proliferative disorders.

BACKGROUND OF THE INVENTION

[0003] Motor neurons directly control muscle activity throughout thebody. Motor neuron fibers that extend from the spinal cord to the muscletransmit neural impulses. Motor neuron cell bodies lie within graymatter, the inner core of the spinal cord. They are confined to theanterior horn, one of three distinct functional regions of gray matter.The motor neuron cell bodies receive signals primarily from neuronscontained in the other two regions of gray matter. These neuronstransmit signals from the brain and other regions of the spinal cord.

[0004] Spinal muscular atrophy (SMA) is a fatal neurodegenerativedisorder that specifically affects motor neurons of the anterior horn.Extensive loss of these neurons results in progressive muscle weaknessand paralysis leading to muscular atrophy. SMA is an autosomal recessivedisorder that occurs once in every 6000 live births and has a carrierfrequency of 1 in 40. Cystic fibrosis is the only fatal autosomalrecessive disorder that occurs with greater frequency. SMA afflictschildren, and three types of SMA have been classified based on age ofonset and clinical course of the disease. Type I, also called infantileSMA or Werdnig-Hoffman disease, is the most severe form with onsetbefore six months of age and death from respiratory failure by two yearsof age. Type II, also called chronic childhood SMA or intermediate SMA,presents at around 18 months of age and progresses slowly. Afflictedchildren cannot walk unaided but survive beyond four years of age. TypeII, also called Wohlfart-Kugelberg-Welander disease, is the mildest formwith onset ranging from two years of age to adolescence and variabledegrees of muscular weakness (Lefebvre et al. (1995) Cell 80:155-165).

[0005] SMA is caused by lesions in the survival motor neuron (smn) geneon chromosome 5q13 (Burglen et al. (1996) Genomics 32:479-482). Thenormal chromosome 5 contains a duplication of the smn locus, resultingin a telomere proximal smn gene (smn^(T)) and a centromere proximal smngene (smn^(C)). The two genes are nearly identical in nucleotidesequence, and both encode a 294-amino acid protein of 38 kilodaltons.However, the smn^(C) RNA transcript can be alternatively spliced, andthe resultant protein is truncated at the C-terminus. The function ofthis alternative protein product is unknown. Molecular genetic analysisindicates that in over 98% of patients with SMA, sma^(T) is completelyor partially deleted. In the remaining 2%, smn^(T) contains pointmutations or alterations in splice site consensus sequences. Inaddition, the severity of the lesion in smn^(T) is correlated with theclinical severity of SMA. These data indicate that smn^(T), and notsmn^(c), plays a critical role in the determination of SMA (Lefebvre,supra). However, some studies indicate that the activity of smn^(c) maymodulate the clinical severity of SMA as previously established bydefects in smn^(T) (Coovert et al. (1997) Hum Mol Genet 6:1205-1214). Ingeneral, detection of lesions in smn^(T) may provide the basis fordefinitive prenatal and childhood diagnosis of SMA.

[0006] Quantitative western analysis shows that the protein, SMN, isnormally expressed at high levels not only in the spinal cord, but alsoin the kidney, liver, and brain. Intermediate SMN levels are detected inskeletal and cardiac muscle, and low levels are detected in primaryfibroblasts and lymphoblasts. The role, if any, for SMN outside of thespinal cord is unclear, as the pathology of SMA is specific to motorneuron muscle control (Coovert, supra). At the cellular level,immunocytochemistry demonstrates that SMN is localized to both thecytoplasm and the nucleus. SMN is diffusely distributed throughout thecytoplasm, while nuclear SMN is concentrated at discrete foci. Thesefoci, called gems, are novel structures that are intimately associatedwith coiled bodies. Coiled bodies are subnuclear structures involved inRNA processing and metabolism. An in vivo screen for SMN-interactingpolypeptides identified fibrillarin, a known component of coiled bodies,and the RGG RNA-binding motif of hnRNP U, a nuclear protein involved inRNA processing. These data suggest that the molecular basis of SMA mayinvolve defects in RNA processing in motor neurons (Liu and Dreyfuss(1996) EMBO J 15:3555-3565).

[0007] The mouse homolog of smn has been cloned and localized tochromosome 13 in a region syntenic to that of human chromosome 5q13.Unlike human smn, mouse smn (msmn) is a single-copy gene, suggestingthat duplication of the human locus is a recent evolutionary event. msmnencodes a 288-amino acid protein that shares 82% amino acid identitywith human smn. Northern analysis shows that msmn RNA is widelyexpressed in various tissues, including heart, brain, kidney and testis(Viollet et al. (1997) Genomics 40:185-188). Homozygous deletion of msmnis lethal during the morula (16-64 cell) stage of embryogenesis. Thisphenotype is much more severe than that of SMA in humans, suggestingthat differences in gene copy number may influence the severity of theSMA phenotype. In humans, smn^(c) may partially compensate for deletionof smn^(T) to delay disease onset and prolong survival (Schrank et al.(1997) Proc Natl Acad Sci 94:9920-9925).

[0008] The discovery of a new human SMN-like protein and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, treatment, andprevention of neurological, reproductive, and cell proliferativedisorders.

SUMMARY OF THE INVENTION

[0009] The invention is based on the discovery of a human SMN-likeprotein, HSLP, which shows homology to mouse and human SMN, a proteininvolved in motor neuron survival. The invention features asubstantially purified protein comprising the amino acid sequence of SEQID NO:1 or a fragment of SEQ ID NO:1.

[0010] The invention further provides a substantially purified varianthaving at least 90% amino acid sequence identity to the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention alsoprovides an isolated and purified polynucleotide encoding the proteincomprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. Theinvention also includes an isolated and purified polynucleotide varianthaving at least 90% polynucleotide sequence identity to thepolynucleotide encoding the protein comprising the amino acid sequenceof SEQ ID NO:1 or a fragment of SEQ ID NO:1.

[0011] The invention further provides an isolated and purifiedpolynucleotide which hybridizes under stringent conditions to thepolynucleotide encoding the protein comprising the amino acid sequenceof SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated andpurified polynucleotide which is complementary to the polynucleotideencoding the protein comprising the amino acid sequence of SEQ ID NO:1or a fragment of SEQ ID NO:1.

[0012] The invention also provides an isolated and purifiedpolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2, and an isolated and purified polynucleotidevariant having at least 90% polynucleotide sequence identity to thepolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2. The invention also provides an isolated andpurified polynucleotide having a sequence complementary to thepolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2. The invention also provides a polynucleotidefragment comprising nucleotides 712-747 for detecting the presence orexpression of an identical endogenous gene.

[0013] The invention further provides an expression vector containing atleast a fragment of the polynucleotide encoding the protein comprisingthe sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In anotheraspect, the expression vector is contained within a host cell.

[0014] The invention also provides a method for producing a proteincomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, the method comprising the steps of: (a) culturing the host cellcontaining an expression vector containing at least a fragment of apolynucleotide encoding the protein comprising the amino acid sequenceof SEQ ID NO:1 or a fragment of SEQ ID NO: 1 under conditions suitablefor the expression of the protein; and (b) recovering the protein fromthe host cell culture.

[0015] The invention also provides a pharmaceutical compositioncomprising a substantially purified protein having the sequence of SEQID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitablepharmaceutical carrier.

[0016] The invention further includes a purified antibody which binds toa protein comprising the sequence of SEQ ID NO:1 or a fragment of SEQ IDNO:1, as well as a purified agonist and a purified antagonist of theprotein.

[0017] The invention also provides a method for treating or preventing aneurological disorder, the method comprising administering to a subjectin need of such treatment an effective amount of a pharmaceuticalcomposition comprising substantially purified protein having the aminoacid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

[0018] The invention also provides a method for treating or preventing areproductive disorder, the method comprising administering to a subjectin need of such treatment an effective amount of a pharmaceuticalcomposition comprising substantially purified protein having the aminoacid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

[0019] The invention also provides a method for treating or preventing acell proliferative disorder, the method comprising administering to asubject in need of such treatment an effective amount of an antagonistof the protein having the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1.

[0020] The invention also provides a method for detecting apolynucleotide encoding a protein comprising the amino acid sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1 in a biological samplecontaining nucleic acids, the method comprising the steps of: (a)hybridizing the complement of the polynucleotide encoding the proteincomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1 to at least one of the nucleic acids of the biological sample,thereby forming a hybridization complex; and (b) detecting thehybridization complex, wherein the presence of the hybridization complexcorrelates with the presence of a polynucleotide encoding the proteincomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1 in the biological sample. In one aspect, the nucleic acids ofthe biological sample are amplified by the polymerase chain reactionprior to the hybridizing step.

BRIEF DESCRIPTION OF THE FIGURES

[0021]FIGS. 1A, 1B, 1C, 1D, 1E, and 1F show the amino acid sequence (SEQID NO:1) and nucleic acid sequence (SEQ ID NO:2) of HSLP. The alignmentwas produced using LASERGENE software (Hitachi Software Engineering,South San Francisco Calif.).

[0022]FIGS. 2A and 2B show the amino acid sequence alignments among HSLP(3769729; SEQ ID NO:1), mouse SMN (GI 1857114; SEQ ID NO:3), and humanSMN (GI 1314346; SEQ ID NO:4), produced using the multisequencealignment program of LASERGENE software (DNASTAR, Madison Wis.).

DESCRIPTION OF THE INVENTION

[0023] Before the present proteins, polynucleotides, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0024] As used herein and in the appended claims, the singular forms“a”, “an”, and “the” include plural reference unless the context clearlydictates otherwise. For example, a reference to “a host cell” includes aplurality of such host cells, and a reference to “an antibody”encompasses one or more antibodies and equivalents thereof known tothose skilled in the art.

[0025] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publications andpatents mentioned herein are incorporated by reference herein and arecited for the purpose of describing and disclosing the cell lines,vectors, and methodologies which are reported in the publications andwhich might be used in connection with the invention. Nothing herein isto be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

[0026] Definitions

[0027] “HSLP” refers to a protein comprising the amino acid sequence ofan SMN-like protein obtained from any species including bovine, ovine,porcine, murine, equine, and preferably the human species, from anysource, whether natural, synthetic, semi-synthetic, or recombinant.

[0028] “Agonist” refers to a molecule which, when bound to HSLP,increases or prolongs the duration of the effect of HSLP. Agonists mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to and modulate the effect of HSLP.

[0029] An “allele” is an alternative form of the gene encoding HSLP.Alleles may result from at least one mutation in the nucleic acidsequence and may result in altered mRNAs or in polypeptides whosestructure or function may or may not be altered. Any given natural orrecombinant gene may have none, one, or many allelic forms. Commonmutational changes which give rise to alleles are generally ascribed tonatural deletions, additions, or substitutions of nucleotides. Each ofthese types of changes may occur alone, or in combination with theothers, one or more times in a given sequence.

[0030] “Altered” nucleic acid sequences encoding HSLP include thosesequences with deletions, insertions, or substitutions of differentnucleotides, resulting in a polynucleotide the same HSLP or apolypeptide with at least one functional characteristic of HSLP.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding HSLP, and improper or unexpected hybridizationto alleles, with a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding HSLP. The encoded protein may also be“altered” and may contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent HSLP. Deliberate amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of HSLP isretained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, positively charged amino acids mayinclude lysine and arginine, and amino acids with uncharged polar headgroups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; and phenylalanine and tyrosine.

[0031] “Amino acid sequence” refers to an oligopeptide, peptide,polypeptide, or protein sequence, or a fragment of any of these, and tonaturally occurring or synthetic molecules. In this context,“fragments”, “immunogenic fragments”, or “antigenic fragments” refer tofragments of HSLP which are preferably about 5 to about 15 amino acidsin length and which retain some biological activity or immunologicalactivity of HSLP. The amino acid sequence is not limited to the completenative amino acid sequence associated with the recited protein molecule.

[0032] “Amplification” relates to the production of additional copies ofa nucleic acid sequence. Amplification is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art.

[0033] “Antagonist” refers to a molecule which, when bound to HSLP,decreases the amount or the duration of the effect of the biological orimmunological activity of HSLP. Antagonists may include proteins,nucleic acids, carbohydrates, antibodies, or any other molecules whichdecrease the effect of HSLP.

[0034] “Antibody” refers to intact molecules as well as to fragmentsthereof, such as Fab, F(ab′)₂, and Fv fragments, which are capable ofbinding the epitopic determinant. Antibodies that bind HSLP polypeptidescan be prepared using intact polypeptides or using fragments containingsmall peptides of interest as the immunizing antigen. The polypeptide oroligopeptide used to immunize an animal (e.g., a mouse, a rat, or arabbit) can be derived from the translation of RNA, or synthesizedchemically, and can be conjugated to a carrier protein if desired.Commonly used carriers that are chemically coupled to peptides includebovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin(KLH). The coupled peptide is then used to immunize the animal.

[0035] “Antigenic determinant” refers to that fragment of a molecule(i.e., an epitope) that makes contact with a particular antibody. When aprotein or a fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodieswhich bind specifically to antigenic determinants (given regions orthree-dimensional structures on the protein). An antigenic determinantmay compete with the intact antigen (i.e., the immunogen used to elicitthe immune response) for binding to an antibody.

[0036] “Antisense” refers to any composition containing a nucleic acidsequence which is complementary to a specific nucleic acid sequence.“Antisense strand” is used in reference to a nucleic acid strand that iscomplementary to the “sense” strand. Antisense molecules may be producedby any method including synthesis or transcription. Once introduced intoa cell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and to block either transcriptionor translation. The designation “negative” can refer to the antisensestrand, and the designation “positive” can refer to the sense strand.

[0037] “Biologically active” refers to a protein having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” refers to the capability of thenatural, recombinant, or synthetic HSLP, or of any oligopeptide thereof,to induce a specific immune response in appropriate animals or cells andto bind with specific antibodies.

[0038] “Complementary” or “complementarity” refer to the natural bindingof polynucleotides under permissive salt and temperature conditions bybase pairing. For example, the sequence “A-G-T” binds to thecomplementary sequence “T-C-A”. Complementarity between twosingle-stranded molecules may be “partial”, such that only some of thenucleic acids bind, or it may be “complete”, such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of the hybridization between the nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands, andin the design and use of peptide nucleic acid (PNA) molecules.

[0039] A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence” refer broadly toany composition containing the given polynucleotide or amino acidsequence. The composition may comprise a dry formulation, an aqueoussolution, or a sterile composition. Compositions comprisingpolynucleotide sequences encoding HSLP or fragments of HSLP may beemployed as hybridization probes. The probes may be stored infreeze-dried form and may be associated with a stabilizing agent such asa carbohydrate. In hybridizations, the probe may be deployed in anaqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS),and other components (e.g., Denhardt's solution, dry milk, salmon spermDNA, and the like).

[0040] “Consensus sequence” refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, extended using XL-PCR kit(PE Biosystems, Foster City Calif.) in the 5′ and/or the 3′ direction,and resequenced, or which has been assembled from the overlappingsequences of more than one Incyte Clone using a computer program forfragment assembly, such as the GELVIEW fragment assembly system(Computer Genetics Group (GCG), Madison, Wis.). Some sequences have beenboth extended and assembled to produce the consensus sequence.

[0041] The phrase “correlates with expression of a polynucleotide”indicates that the detection of the presence of nucleic acids, the sameor related to a nucleic acid sequence encoding HSLP, by northernanalysis is indicative of the presence of nucleic acids encoding HSLP ina sample, and thereby correlates with expression of the transcript fromthe polynucleotide encoding HSLP.

[0042] A “deletion” refers to a change in the amino acid or nucleotidesequence that results in the absence of one or more amino acid residuesor nucleotides.

[0043] “Derivative” refers to the chemical modification of HSLP, of apolynucleotide sequence encoding HSLP, or of a polynucleotide sequencecomplementary to a polynucleotide sequence encoding HSLP. Chemicalmodifications of a polynucleotide sequence can include, for example,replacement of hydrogen by an alkyl, acyl, or amino group. A derivativepolynucleotide encodes a polypeptide which retains at least onebiological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

[0044] “Homology” refers to a degree of complementarity. A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to as“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization, and the like) under conditions of reduced stringency.

[0045] “Hybridization stringency” is determined by G+C content of theprobe, salt concentration, and temperature. In particular, stringencycan be increased by reducing the concentration of salt or raising thehybridization temperature. In solutions used for some substrate basedhybridizations, additions of an organic solvent such as formamide allowsthe reaction to occur at a lower temperature. Hybridization can beperformed at low stringency with buffers, such as 5×SSC with 1% sodiumdodecyl sulfate (SDS) at 60° C., which permits the formation of ahybridization complex between nucleotide sequences that contain somemismatches. Subsequent washes are performed at higher stringency withbuffers such as 0.2×SSC with 0.1% SDS at either 45° C. (mediumstringency) or 68° C. (high stringency). At high stringency,hybridization complexes will remain stable only where the nucleic acidsequences are completely complementary. In some membrane-basedhybridizations, perferably 35% or most preferably 50%, formamide can beadded to the hybridization solution to reduce the temperature at whichhybridization is performed, and background signals can be reduced by theuse of other detergents such as Sarkosyl or Triton X-100 and a blockingagent such as salmon sperm DNA. Selection of components and conditionsfor hybridization are well known to those skilled in the art.

[0046] “Percent identity” refers to the percentage of sequencesimilarity found in a comparison of two or more amino acid or nucleicacid sequences. Percent identity can be determined electronically, e.g.,by using the MEGALIGN program (DNASTAR, Madison, Wis.). This program cancreate alignments between two or more sequences according to differentmethods, e.g., the clustal method. (See, e.g., Higgins and Sharp (1988)Gene 73:237-244.) The clustal algorithm groups sequences into clustersby examining the distances between all pairs. The clusters are alignedpairwise and then in groups. The percentage similarity between two aminoacid sequences, e.g., sequence A and sequence B, is calculated bydividing the length of sequence A, minus the number of gap residues insequence A, minus the number of gap residues in sequence B, into the sumof the residue matches between sequence A and sequence B, times onehundred. Gaps of low or of no homology between the two amino acidsequences are not included in determining percentage similarity.

[0047] “Human artificial chromosomes” (HACs) are linear microchromosomeswhich may contain DNA sequences of about 6 kb to 10 Mb in size, andwhich contain all of the elements required for stable mitotic chromosomesegregation and maintenance.

[0048] “Humanized antibody” refers to antibody molecules in which theamino acid sequence in the non-antigen binding regions has been alteredso that the antibody more closely resembles a human antibody, and stillretains its original binding ability.

[0049] “Hybridization” refers to any process by which a strand ofnucleic acid binds with a complementary strand through base pairing.“Hybridization complex” refers to a complex formed between two nucleicacid sequences by virtue of the formation of hydrogen bonds betweencomplementary bases. A hybridization complex may be formed in solution(e.g., C₀t or R₀t analysis) or formed between one nucleic acid sequencepresent in solution and another nucleic acid sequence immobilized on asubstrate (e.g., paper, membranes, filters, chips, pins or glass slides,or any other appropriate substrate to which cells or their nucleic acidshave been fixed).

[0050] “Insertion” and “addition” refer to changes in an amino acid ornucleotide sequence resulting in the addition of one or more amino acidresidues or nucleotides, respectively, to the sequence found in thenaturally occurring molecule.

[0051] “Immune response” refers to conditions associated withinflammation, trauma, immune disorders, or infectious or geneticdisease, and the like. These conditions can be characterized byexpression of various factors, e.g., cytokines, chemokines, and othersignaling molecules, which may affect cellular and systemic defensesystems.

[0052] “Microarray” refers to an arrangement of distinct polynucleotideson a substrate.

[0053] “Element” and “array element” refer to a hybridizablepolynucleotide arrayed on the surface of a microarray.

[0054] “Modulate” refers to a change in the activity of HSLP. Forexample, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of HSLP.

[0055] “Nucleic acid sequence” refers to an oligonucleotide, nucleotide,polynucleotide, or any fragment thereof, to DNA or RNA of genomic orsynthetic origin (cDNA) which may be single-stranded or double-strandedand may represent the sense or the antisense strand, to peptide nucleicacid (PNA), or to any DNA-like or RNA-like material. In this context,“fragments” refer to those nucleic acid sequences which are greater thanabout 60 nucleotides in length, and most preferably are at least about100 nucleotides, at least about 1000 nucleotides, or at least about10,000 nucleotides in length.

[0056] The terms “operably associated” or “operably linked” refer tofunctionally related nucleic acid sequences. A promoter is operablyassociated or operably linked with a coding sequence if the promotercontrols the transcription of the encoded polypeptide. While operablyassociated or operably linked nucleic acid sequences can be contiguousand in reading frame, certain genetic elements, e.g., repressor genes,are not contiguously linked to the encoded polypeptide but still bind tooperator sequences that control expression of the polypeptide.

[0057] “Oligonucleotide” refers to a nucleic acid sequence of at leastabout 6 nucleotides to 60 nucleotides, preferably about 15 to 30nucleotides, and most preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or in a hybridization assay or microarray.Oligonucleotide is substantially equivalent to the terms “amplimer,”“primer,” “oligomer,” and “probe,” as these terms are commonly definedin the art.

[0058] “Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAand RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

[0059] The term “sample” is used in its broadest sense. A samplesuspected of containing nucleic acids encoding HSLP, or fragmentsthereof, or HSLP itself, may comprise a bodily fluid; an extract from acell, chromosome, organelle, or membrane isolated from a cell; a cell;genomic DNA, RNA, or cDNA, in solution or bound to a solid support; atissue; a tissue print; a fingerprint, and the like.

[0060] “Specific binding” refers to that interaction between a proteinor peptide and an agonist, an antibody, or an antagonist. Theinteraction is dependent upon the presence of a particular structure ofthe protein, e.g., the antigenic determinant or epitope, recognized bythe binding molecule. For example, if an antibody is specific forepitope “A”, the presence of a polypeptide containing the epitope A, orthe presence of free unlabeled A, in a reaction containing free labeledA and the antibody will reduce the amount of labeled A that binds to theantibody.

[0061] “Stringent conditions” refers to conditions which permithybridization between polynucleotide sequences and the claimedpolynucleotide sequences. Suitably stringent conditions can be definedby GC content of the polynucleotide sequence, salt concentration in theprehybridization and hybridization solutions, and hybridizationtemperature. These conditions are well known in the art as is the factthat stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature.

[0062] “Substantially purified” refers to nucleic acid or amino acidsequences that are removed from their natural environment and are mostpreferably about 90% free from other components with which they arenaturally associated.

[0063] A “substitution” refers to the replacement of one or more aminoacids or nucleotides by different amino acids or nucleotides,respectively.

[0064] “Transformation” describes a process by which exogenous DNAenters and changes a recipient cell. Transformation may occur undernatural or artificial conditions according to various methods well knownin the art, and may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method for transformation is selected based on the type ofhost cell being transformed and may include, but is not limited to,viral infection, electroporation, heat shock, lipofection, and particlebombardment. The term “transformed” cells includes stably transformedcells in which the inserted DNA is capable of replication either as anautonomously replicating plasmid or as part of the host chromosome, aswell as transiently transformed cells which express the inserted DNA orRNA for limited periods of time.

[0065] “Variant” refers to an amino acid sequence that is altered by oneor more amino acids. The variant may have “conservative” changes,wherein a substituted amino acid has similar structural or chemicalproperties (e.g., replacement of leucine with isoleucine) or“nonconservative” changes wherein the substituted amino acid isstructurally or chemically different (e.g., replacement of glycine withtryptophan). Guidance in determining which amino acid residues may besubstituted, inserted, or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, LASERGENE software (DNASTAR).

[0066] The Invention

[0067] The invention is based on the discovery of a new human SMN-likeprotein (HSLP), the polynucleotides encoding HSLP, and the use of thesecompositions for the diagnosis, treatment, or prevention ofneurological, reproductive, and cell proliferative disorders.

[0068] Nucleic acids encoding the HSLP of the present invention werefirst identified in Incyte Clone 3769729 from the breast tissue cDNAlibrary (BRSTNOT24) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:2, was derived from thefollowing overlapping and/or extended sequences: Incyte Clones 3769729(BRSTNOT24), 637394 (NEUTGMT01), 2207558 (SINTFET03), 1643342(HEARFET01), and 1272275 (TESTTUT02). A fragment of SEQ ID NO:2 fromabout nucleotide 712 to about nucleotide 747 is useful fordistinguishing nucleotide sequences encoding HSLP from those encodingother known SMN-like proteins. Northern analysis shows the expression ofthis sequence in various libraries, at least 67% of which are associatedwith cell proliferation. In particular, 38% of libraries expressing HSLPare derived from reproductive tissue.

[0069] In one embodiment, the invention encompasses a protein comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A-1F. HSLP is238 amino acids in length and has a potential N-glycosylation site atN₁₀₁; a potential casein kinase II phosphorylation site at S₁₄₁; andfive potential protein kinase C phosphorylation sites at S₁₁, S₇₂, S₁₄₁,T₂₀₆, and T₂₂₇. As shown in FIGS. 2A and 2B, HSLP has chemical andstructural homology with SMN from mouse (GI 1857114) and from human (GI1314346). In particular, HSLP and mouse SMN share 18% identity, and HSLPand human SMN share 17% identity. In addition, the regions of HSLP fromW₇₃ to D₉₈, and from G₁₁₀ to E₁₂₇ are highly conserved among SMNproteins from three divergent mammalian species: human, mouse, and dog.For example, these two regions of HSLP share 73% and 47% identity,respectively, with the homologous regions of mouse and human SMN. HSLPis similar in size to mouse and human SMN which are 288 and 294 aminoacids in length, respectively.

[0070] The invention also encompasses HSLP variants. A preferred HSLPvariant is one which has at least about 80%, more preferably at leastabout 90%, and most preferably at least about 95% amino acid sequenceidentity to the HSLP amino acid sequence, and which contains at leastone functional or structural characteristic of HSLP.

[0071] The invention also encompasses polynucleotides with a deletion,insertion, or substitution but which encodes HSLP or at least onefunctional domain of HSLP. The protein produced by an altered

[0072] The invention also encompasses a variant of a polynucleotidesequence encoding HSLP. In particular, such a variant polynucleotidesequence will have at least about 80%, more preferably at least about90%, and most preferably at least about 95% polynucleotide sequenceidentity to the polynucleotide sequence encoding HSLP. A particularaspect of the invention encompasses a variant of SEQ ID NO:2 which hasat least about 80%, more preferably at least about 90%, and mostpreferably at least about 95% polynucleotide sequence identity to SEQ IDNO:2. Any one of the polynucleotide variants described above can encodean amino acid sequence which contains at least one functional orstructural characteristic of HSLP.

[0073] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude ofpolynucleotide sequences encoding HSLP, some bearing minimal homology tothe polynucleotide sequences of any known and naturally occurring gene,may be produced. Thus, the invention contemplates each and everypossible variation of polynucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the polynucleotide sequence of naturally occurringHSLP, and all such variations are to be considered as being specificallydisclosed.

[0074] Although nucleotide sequences which encode HSLP and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HSLP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HSLP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding HSLP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0075] The invention also encompasses production of DNA sequences whichencode HSLP and HSLP derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding HSLP or any fragment thereof.

[0076] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed polynucleotide sequences,and, in particular, to those shown in SEQ ID NO:2, or a fragment of SEQID NO:2, under various conditions of stringency. (See, e.g., Wahl andBerger (1987) Methods Enzymol 152:399-407, and Kimmel (1987) MethodsEnzymol 152:507-511.)

[0077] Methods for DNA sequencing are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, T7 SEQUENASE DNA polymerase, Taq DNA polymerase, andTHERMOSEQUENASE DNA polymerase (Amersham Pharmacia Biotech (APB),Picataway N.J.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification system(Life Technologies, Gaithersburg Md.). Preferably, the process isautomated with machines such as the MICROLAB 2200 system (Hamilton, RenoNev.), DNA ENGINE thermal cycler (MJ Research, Watertown Mass.), and theABI CATALYST thermal cycler and ABI PRISM 373 and 377 DNA sequencingsystems (PE Biosytems).

[0078] The nucleic acid sequences encoding HSLP may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus. (See, e.g., Sarkar (1993) PCRMethods Applic 2:318-322.) In particular, genomic DNA is first amplifiedin the presence of a primer which is complementary to a linker sequencewithin the vector and a primer specific to a region of the nucleotidesequenc. The amplified sequences are subjected to a second round of PCRwith the same linker primer and another specific primer internal to thefirst one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0079] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region. (See, e.g., Triglia et al.(1988) Nucleic Acids Res 16:8186.) The primers may be designed usingcommercially available software such as OLIGO 4.06 software (NationalBiosciences, Plymouth Minn.) or another appropriate program to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the target sequence at temperatures of about 68°C. to 72° C. The method uses several restriction enzymes to generate asuitable fragment in the known region of a gene. The fragment is thencircularized by intramolecular ligation and used as a PCR template.

[0080] Another method which may be used is capture PCR, which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA. (See, e.g., Lagerstrom et al.(1991) PCR Methods Applic 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to place anengineered double-stranded sequence into an unknown fragment of the DNAmolecule before performing PCR. Other methods which may be used toretrieve unknown sequences are known in the art. (See, e.g., Parker etal. (1991) Nucleic Acids Res 19:3055-3060.) Additionally, one may usePCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo AltoCalif.) to walk genomic DNA. This process avoids the need to screenlibraries and is useful in finding intron/exon junctions.

[0081] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will include moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5′ non-transcribedregulatory regions.

[0082] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and a charge coupled device camera for detection of theemitted wavelengths. Output/light intensity may be converted toelectrical signal using appropriate software (e.g., GENOTYPER andSEQUENCE NAVIGATOR software, PE Biosystems), and the entire process fromloading of samples to computer analysis and electronic data display maybe computer controlled. Capillary electrophoresis is especiallypreferable for the sequencing of small pieces of DNA which might bepresent in limited amounts in a particular sample.

[0083] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode HSLP may be used in recombinant DNAmolecules to direct expression of HSLP, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressHSLP.

[0084] As will be understood by those of skill in the art, it may beadvantageous to produce HSLP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

[0085] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterHSLP-encoding sequences for a variety of reasons including, but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

[0086] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HSLP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of HSLP activity, it may be useful toencode a chimeric HSLP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the HSLP encoding sequence and theheterologous protein sequence, so that HSLP may be cleaved and purifiedaway from the heterologous moiety.

[0087] In another embodiment, sequences encoding HSLP may besynthesized, in whole or in part, using chemical methods well known inthe art. (See, e.g., Caruthers et al. (1980) Nucleic Acids Symp. Ser.7:215-223, and Horn et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.)Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of HSLP, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques. (See, e.g., Roberge et a. (1995) Science269:202-204.) Automated synthesis maybe achieved using the ABI 431Apeptide synthesizer (PE Biosystems). Additionally, the amino acidsequence of HSLP, or any part thereof, may be altered during directsynthesis and/or combined with sequences from other proteins, or anypart thereof, to produce a variant protein.

[0088] The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez and Regnier (1990)Methods Enzymol 182:392-421.) The composition of the synthetic peptidesmay be confirmed by amino acid analysis or by sequencing. (See, e.g.,Creighton (1983) Proteins, Structures and Molecular Properties, W HFreeman, New York N.Y.)

[0089] In order to express a biologically active HSLP, the nucleotidesequences encoding HSLP or derivatives thereof may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0090] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding HSLPand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook etal. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview N.Y., ch. 4, 8, and 16-17; and Ausubel et al. (1995)Current Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., ch. 9, 13, and 16.)

[0091] A variety of expression vector/host systems and control elementsmay be utilized to contain and express sequences encoding HSLP. The“control elements” or “regulatory sequences” are those non-translatedregions, e.g., enhancers, promoters, and 5′ and 3′untranslated regions,of the vector and polynucleotide sequences encoding HSLP which interactwith host cellular proteins to carry out transcription and translation.Such elements may vary in their strength and specificity. Depending onthe vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters, e.g., hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla Calif.) or PSPORT1 plasmid(Life Technologies), may be used. The baculovirus polyhedrin promotermay be used in insect cells. Promoters or enhancers derived from thegenomes of plant cells (e.g., heat shock, RUBISCO, and storage proteingenes) or from plant viruses (e.g., viral promoters or leader sequences)may be cloned into the vector. In mammalian cell systems, promoters frommammalian genes or from mammalian viruses are preferable. If it isnecessary to generate a cell line that contains multiple copies of thesequence encoding HSLP, vectors based on SV40 or EBV may be used with anappropriate selectable marker.

[0092] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for HSLP. For example, whenlarge quantities of HSLP are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, multifunctional E. coli cloning and expression vectors such asPBLUESCRIPT phagemid (Stratagene), in which the sequence encoding HSLPmay be ligated into the vector in frame with sequences for theamino-terminal Met and the subsequent 7 residues of β-galactosidase sothat a hybrid protein is produced, and pIN vectors (Van Heeke andSchuster (1989) J Biol Chem 264:5503-5509). PGEX vectors (APB) may alsobe used to express foreign proteins as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems may be designed to includeheparin, thrombin, or factor XA protease cleavage sites so that thecloned protein of interest can be released from the GST moiety at will.

[0093] In the yeast Saccharomvces cerevisiae, a number of vectorscontaining constitutive or inducible promoters, such as alpha factor,alcohol oxidase, and PGH, may be used. (See, e.g., Ausubel, supra; andGrant et al., (1987) Methods Enzymol 153:516-544.)

[0094] In cases where plant expression vectors are used, the expressionof sequences encoding HSLP may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi et al. (1984) EMBO J.3:1671-1680; Broglie et al. (1984) Science 224:838-843; and Winter etal. (1991) Results Probl Cell Differ 17:85-105). These constructs can beintroduced into plant cells by direct DNA or pathogen-mediatedtransformation. Such techniques are described in a number of availablereviews. (See, e.g., Hobbs or Murry (1992) In: Yearbook of Science andTechnology, McGraw Hill, N.Y. N.Y.; pp. 191-196.)

[0095] An insect system may also be used to express HSLP. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding HSLPmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of sequences encoding HSLP will render thepolyhedrin gene inactive and produce recombinant virus lacking coatprotein. The recombinant viruses may then be used to infect, forexample, S. frugiperda cells or Trichoplusia larvae in which HSLP may beexpressed. (See, e.g., Engelhard et al. (1994) Proc Natl Acad Sci91:3224-3227.)

[0096] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding HSLP may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing HSLP in infected host cells. (See, e.g.,Logan and Shenk (1984) Proc Natl Acad Sci 81:3655-3659.) In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0097] HACs may also be employed to deliver larger fragments of DNA thancan be contained and expressed in a plasmid. HACs of about 6 kb to 10 Mbare constructed and delivered via conventional delivery methods(liposomes, polycationic amino polymers, or vesicles) for therapeuticpurposes.

[0098] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding HSLP. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding HSLP and its initiation codon and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate for the particularcell system used. (See, e.g., Scharf et al. (1994) Results Probl CellDiffer 20:125-162.)

[0099] In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of theprotein include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding,and/or function. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available fromthe American Type Culture Collection (Manassas Va.) and may be chosen toensure the correct modification and processing of the foreign protein.

[0100] For long term, high yield production of recombinant proteins,stable expression is preferred. For example, cell lines capable ofstably expressing HSLP can be transformed using expression vectors whichmay contain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for about 1 to 2 days in enriched media before being switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type. The invention is notlimited by the vector, host cell or control elements employed.

[0101] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase genes and adeninephosphoribosyltransferase genes, which can be employed in tk or aprcells, respectively. (See, e.g., Wigler et al. (1977) Cell 11:223-232,and Lowy et al. (1980) Cell 22:817-823) Also, antimetabolite,antibiotic, or herbicide resistance can be used as the basis forselection. For example, dhfr confers resistance to methotrexate; nptconfers resistance to the aminoglycosides neomycin and G-418; and alsand pat confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively. (See, e.g., Wigler et al. (1980) ProcNatl Acad Sci 77:3567-3570, Colbere-Garapin et al. (1981) J Mol Biol150:1-14, and Murry, supra.) Additional selectable genes have beendescribed, e.g., trpB, which allows cells to utilize indole in place oftryptophan, or hisD, which allows cells to utilize histinol in place ofhistidine. (See, e.g., Hartman and Mulligan (1988) Proc Natl Acad Sci85:8047-8051.) Visible markers, e.g., anthocyanins, B glucuronidase andits substrate GUS, luciferase and its substrate luciferin may be used.Green fluorescent proteins (GFP; Clontech) can also be used. Thesemarkers can be used not only to identify transformants, but also toquantify the amount of transient or stable protein expressionattributable to a specific vector system. (See, e.g., Rhodes et al.(1995) Methods Mol Biol 55:121-131.)

[0102] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, the presence and expressionof the gene may need to be confirmed. For example, if the sequenceencoding HSLP is inserted within a marker gene sequence, transformedcells containing sequences encoding HSLP can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding HSLP under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

[0103] Alternatively, host cells which contain the nucleic acid sequenceencoding HSLP and express HSLP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein sequences.

[0104] The presence of polynucleotide sequences encoding HSLP can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding HSLP.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding HSLP todetect transformants containing DNA or RNA encoding HSLP.

[0105] A variety of protocols for detecting and measuring the expressionof HSLP, using either polyclonal or monoclonal antibodies specific forthe protein, are known in the art. Examples of such techniques includeenzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on HSLP is preferred, but a competitivebinding assay may be employed. These and other assays are well describedin the art. (See, e.g., Hampton et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul Minn., Section IV; and Maddox etal. (1983) J Exp Med 158:1211-1216).

[0106] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding HSLPinclude oligolabeling, nick translation, end-labeling, or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding HSLP, or any fragments thereof, may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided by ABPand Promega (Madison Wis.). Suitable reporter molecules or labels whichmay be used for ease of detection include radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents, as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

[0107] Host cells transformed with nucleotide sequences encoding HSLPmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode HSLP may be designed to contain signal sequences which directsecretion of HSLP through a prokaryotic or eukaryotic cell membrane.Other constructions may be used to join sequences encoding HSLP tonucleotide sequences encoding a protein domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAG extension/affinitypurification system (Immunex, Seattle Wash.). The inclusion of cleavablelinker sequences, such as those specific for Factor XA or enterokinase(Invitrogen, San Diego Calif.), between the purification domain and theHSLP encoding sequence may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingHSLP and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on immobilized metal ion affinity chromatography(IMIAC; Porath et al. (1992) Prot Exp Purif 3:263-281). The enterokinasecleavage site provides a means for purifying HSLP from the fusionprotein (Kroll et al. (1993) DNA Cell Biol 12:441-453).

[0108] Fragments of HSLP may be produced not only by recombinantproduction, but also by direct peptide synthesis using solid-phasetechniques. (See, e.g., Creighton (1984) Protein: Structures andMolecular Properties, W H Freeman, New York N.Y., pp. 55-60.) Proteinsynthesis may be performed by manual techniques or by automation.Automated synthesis may be achieved, for example, using the ABI 431Apeptide synthesizer (PE Biosystems). Various fragments of HSLP may besynthesized separately and then combined to produce the full lengthmolecule.

[0109] Therapeutics

[0110] Chemical and structural homology exists among HSLP and SMN frommouse (GI 1857114) and human (GI 1314346). In addition, HSLP isexpressed in reproductive and proliferating tissues. Therefore, HSLPappears to play a role in neurological, reproductive, and cellproliferative disorders.

[0111] Therefore, in one embodiment, HSLP or a fragment or derivativethereof may be administered to a subject to treat or prevent aneurological disorder. Such disorders can include, but are not limitedto, akathesia, Alzheimer's disease, amnesia, amyotrophic lateralsclerosis, bipolar disorder, catatonia, cerebral neoplasms, dementia,depression, diabetic neuropathy, Down's syndrome, tardive dyskinesia,dystonias, epilepsy, Huntington's disease, peripheral neuropathy,multiple sclerosis, neurofibromatosis, Parkinson's disease, paranoidpsychoses, postherpetic neuralgia, schizophrenia, and Tourette'sdisorder.

[0112] In another embodiment, a vector capable of expressing HSLP or afragment or derivative thereof may be administered to a subject to treator prevent a neurological disorder including, but not limited to, thosedescribed above.

[0113] In a further embodiment, a pharmaceutical composition comprisinga substantially purified HSLP in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a neurological disorder including, but not limited to, thoseprovided above.

[0114] In still another embodiment, an agonist which modulates theactivity of HSLP may be administered to a subject to treat or prevent aneurological disorder including, but not limited to, those listed above.

[0115] In another embodiment, HSLP or a fragment or derivative thereofmay be administered to a subject to treat or prevent a reproductivedisorder. Such disorders can include, but are not limited to, abnormalprolactin production, infertility, tubal disease, ovulatory defects,endometriosis, perturbations of the estrous and menstrual cycles,polycystic ovary syndrome, ovarian hyperstimulation syndrome,endometrial and ovarian tumors, autoimmune disorders, ectopic pregnancy,teratogenesis, breast cancer, fibrocystic breast disease, galactorrhea,abnormal spermatogenesis, abnormal sperm physiology, testicular cancer,prostate cancer, benign prostatic hyperplasia, prostatitis, andgynecomastia.

[0116] In another embodiment, a vector capable of expressing HSLP or afragment or derivative thereof may be administered to a subject to treator prevent a reproductive disorder including, but not limited to, thosedescribed above.

[0117] In a further embodiment, a pharmaceutical composition comprisinga substantially purified HSLP in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a reproductive disorder including, but not limited to, thoseprovided above.

[0118] In still another embodiment, an agonist which modulates theactivity of HSLP may be administered to a subject to treat or prevent areproductive disorder including, but not limited to, those listed above.

[0119] In a further embodiment, an antagonist of HSLP may beadministered to a subject to treat or prevent a cell proliferativedisorder. Such disorders may include, but are not limited to,arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixedconnective tissue disease, myelofibrosis, paroxysmal nocturnalhemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia,and cancers including adenocarcinoma, leukemia, lymphoma, melanoma,myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of theadrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,muscle, ovary, nerve, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus. In oneaspect, an antibody which specifically binds HSLP may be used directlyas an antagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express HSLP.

[0120] In an additional embodiment, a vector expressing the complementof the polynucleotide encoding HSLP may be administered to a subject totreat or prevent a cell proliferative disorder including, but notlimited to, those described above.

[0121] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences, or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0122] An antagonist of HSLP may be produced using methods which aregenerally known in the art. In particular, purified HSLP may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind HSLP. Antibodies to HSLP may alsobe generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, and single chain antibodies, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0123] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith HSLP or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

[0124] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to HSLP have an amino acid sequence consistingof at least about 5 amino acids, and, more preferably, of at least about10 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are identical to a portion of the amino acidsequence of the natural protein and contain the entire amino acidsequence of a small, naturally occurring molecule. Short stretches ofHSLP amino acids may be fused with those of another protein, such asKLH, and antibodies to the chimeric molecule may be produced.

[0125] Monoclonal antibodies to HSLP may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (See, e.g., Kohler et al. (1975) Nature256:495-497, Kozbor et al. (1985) J Immunol Methods 81:31-42, Cote etal. (1983) Proc Natl Acad Sci 80:2026-2030, and Cole et al. (1984) MolCell Biol 62:109-120.)

[0126] In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison et al. (1984)Proc Natl Acad Sci 81:6851-6855, Neuberger et al. (1984) Nature312:604-608, and Takeda et al. (1985) Nature 314:452-454.)Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceHSLP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries.(See, e.g., Burton (1991) Proc Natl Acad Sci 88:10134-10137.)

[0127] Antibodies may also be produced by inducing n vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature. (See, e.g., Orlandi et al. (1989) Proc Natl Acad Sci 86:3833-3837, and Winter et al. (1991) Nature 349:293-299.)

[0128] Antibody fragments which contain specific binding sites for HSLPmay also be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huseet al. (1989) Science 246:1275-1281.)

[0129] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between HSLP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HSLP epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

[0130] In another embodiment of the invention, the polynucleotidesencoding HSLP, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding HSLP may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding HSLP. Thus, complementary molecules or fragments may be used tomodulate HSLP activity, or to achieve regulation of gene function. Suchtechnology is now well known in the art, and sense or antisenseoligonucleotides or larger fragments can be designed from variouslocations along the coding or control regions of sequences encodingHSLP.

[0131] Expression vectors derived from retroviruses, adenoviruses, orherpes or vaccinia viruses, or from various bacterial plasmids, may beused for delivery of nucleotide sequences to the targeted organ, tissue,or cell population. Methods which are well known to those skilled in theart can be used to construct vectors which will express nucleic acidsequences complementary to the polynucleotides of the gene encodingHSLP. (See, e.g., Sambrook, supra; and Ausubel, supra.)

[0132] Genes encoding HSLP can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide, or fragment thereof, encoding HSLP. Such constructs maybe used to introduce untranslatable sense or antisense sequences into acell. Even in the absence of integration into the DNA, such vectors maycontinue to transcribe RNA molecules until they are disabled byendogenous nucleases. Transient expression may last for a month or morewith a non-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

[0133] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′, or regulatory regions of the geneencoding HSLP. Oligonucleotides derived from the transcriptioninitiation site, e.g., between about positions −10 and +10 from thestart site, are preferred. Similarly, inhibition can be achieved usingtriple helix base-pairing methodology. Triple helix pairing is usefulbecause it causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee et al. (1994) In:Huber and Carr, Molecular and Immunologic Approaches, Futura Publishing,Mt. Kisco N.Y., pp. 163-177.) A complementary sequence or antisensemolecule may also be designed to block translation of mRNA by preventingthe transcript from binding to ribosomes.

[0134] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingHSLP.

[0135] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0136] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding HSLP. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA, constitutively or inducibly, can be introduced into cell lines,cells, or tissues.

[0137] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule, or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0138] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposomeinjections, or by polycationic amino polymers may be achieved usingmethods which are well known in the art. (See, e.g., Goldman et al.(1997) Nature Biotechnol 15:462-466.)

[0139] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0140] An additional embodiment of the invention relates to theadministration of a pharmaceutical or sterile composition, inconjunction with a pharmaceutically acceptable carrier, for any of thetherapeutic effects discussed above. Such pharmaceutical compositionsmay consist of HSLP, antibodies to HSLP, and mimetics, agonists,antagonists, or inhibitors of HSLP. The compositions may be administeredalone or in combination with at least one other agent, such as astabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,drugs, or hormones.

[0141] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0142] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing, Easton Pa.).

[0143] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0144] Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

[0145] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0146] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0147] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also contain suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

[0148] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0149] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0150] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acids. Saltstend to be more soluble in aqueous or other protonic solvents than arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

[0151] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of HSLP, such labeling wouldinclude amount, frequency, and method of administration.

[0152] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0153] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells or in animal models such as mice, rats, rabbits, dogs, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0154] A therapeutically effective dose refers to that amount of activeingredient, for example HSLP or fragments thereof, antibodies of HSLP,and agonists, antagonists or inhibitors of HSLP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED50 (the dosetherapeutically effective in 50% of the population) or LD50 (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe LD50/ED50 ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED50 withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

[0155] The exact dosage will be determined by the practitioner, in lightof factors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

[0156] Normal dosage amounts may vary from about 0.1 μg to 100,000 μg,up to a total dose of about 1 gram, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or proteins will be specific toparticular cells, conditions, locations, and the like

[0157] Diagnostics

[0158] In another embodiment, antibodies which specifically bind HSLPmay be used for the diagnosis of disorders characterized by expressionof HSLP, or in assays to monitor patients being treated with HSLP oragonists, antagonists, or inhibitors of HSLP. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for HSLP include methods whichutilize the antibody and a label to detect HSLP in human body fluids orin extracts of cells or tissues. The antibodies may be used with orwithout modification, and may be labeled by covalent or non-covalentattachment of a reporter molecule. A wide variety of reporter molecules,several of which are described above, are known in the art and may beused.

[0159] A variety of protocols for measuring HSLP, including ELISAs,RIAs, and FACS, are known in the art and provide a basis for diagnosingaltered or abnormal levels of HSLP expression. Normal or standard valuesfor HSLP expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to HSLP under conditions suitable for complex formation. Theamount of standard complex formation may be quantitated by variousmethods, preferably by photometric means. Quantities of HSLP expressedin subject, control, and disease samples from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0160] In another embodiment of the invention, the polynucleotidesencoding HSLP may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof HSLP may be correlated with disease. The diagnostic assay may be usedto determine absence, presence, and excess expression of HSLP, and tomonitor regulation of HSLP levels during therapeutic intervention.

[0161] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HSLP or closely related molecules may be used to identifynucleic acid sequences which encode HSLP. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatoryregion, or from a less specific region, e.g., a conserved motif, and thestringency of the hybridization or amplification (maximal, high,intermediate, or low), will determine whether the probe identifies onlynaturally occurring sequences encoding HSLP, alleles, or relatedsequences.

[0162] Probes may also be used for the detection of related sequences,and should preferably have at least 50% sequence identity to any of theHSLP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:2 or from genomic sequences including promoters, enhancers, andintrons of the HSLP gene.

[0163] Means for producing specific hybridization probes for DNAsencoding HSLP include the cloning of polynucleotide sequences encodingHSLP or HSLP derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by means of the addition ofthe appropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

[0164] Polynucleotide sequences encoding HSLP may be used for thediagnosis of a disorder associated with expression of HSLP. Examples ofsuch a disorder include, but are not limited to, a neurological disordersuch as akathesia, Alzheimer's disease, amnesia, amyotrophic lateralsclerosis, bipolar disorder, catatonia, cerebral neoplasms, dementia,depression, diabetic neuropathy, Down's syndrome, tardive dyskinesia,dystonias, epilepsy, Huntington's disease, peripheral neuropathy,multiple sclerosis, neurofibromatosis, Parkinson's disease, paranoidpsychoses, postherpetic neuralgia, schizophrenia, and Tourette'sdisorder; a reproductive disorder such as abnormal prolactin production,infertility, tubal disease, ovulatory defects, endometriosis,perturbations of the estrous and menstrual cycles, polycystic ovarysyndrome, ovarian hyperstimulation syndrome, endometrial and ovariantumors, autoimmune disorders, ectopic pregnancy, teratogenesis, breastcancer, fibrocystic breast disease, galactorrhea, abnormalspermatogenesis, abnormal sperm physiology, testicular cancer, prostatecancer, benign prostatic hyperplasia, prostatitis, and gynecomastia; anda cell proliferative disorder such as arteriosclerosis, atherosclerosis,bursitis, cirrhosis, hepatitis, mixed connective tissue disease,myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,psoriasis, primary thrombocythemia, and cancers includingadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, nerve, pancreas, parathyroid, penis, prostate, salivary glands,skin, spleen, testis, thymus, thyroid, and uterus. The polynucleotidesequences encoding HSLP may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; indipstick, pin, and ELISA-like assays; and in microarrays utilizingfluids or tissues from patients to detect altered HSLP expression. Suchqualitative or quantitative methods are well known in the art.

[0165] In a particular aspect, the nucleotide sequences encoding HSLPmay be useful in assays that detect the presence of associateddisorders, particularly those mentioned above. The nucleotide sequencesencoding HSLP may be labeled by standard methods and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantitated and compared with astandard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of nucleotide sequences encoding HSLP in thesample indicates the presence of the associated disorder. Such assaysmay also be used to evaluate the efficacy of a particular therapeutictreatment regimen in animal studies, in clinical trials, or to monitorthe treatment of an individual patient.

[0166] In order to provide a basis for the diagnosis of a disorderassociated with expression of HSLP, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, encoding HSLP, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with values from an experiment in which a known amountof a substantially purified polynucleotide is used. Standard valuesobtained in this manner may be compared with values obtained fromsamples from patients who are symptomatic for a disorder. Deviation fromstandard values is used to establish the presence of a disorder.

[0167] Once the presence of a disorder is established and a treatmentprotocol is initiated, hybridization assays may be repeated on a regularbasis to determine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0168] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0169] Additional diagnostic uses for oligonucleotides designed from thesequences encoding HSLP may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding HSLP, or a fragment of a polynucleotide complementary to thepolynucleotide encoding HSLP, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

[0170] Methods which may also be used to quantitate the expression ofHSLP include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and interpolating results from standardcurves. (See, e.g., Melby et al. (1993) J Immunol Methods 159:235-244,and Duplaa et al. (1993) Anal Biochem 212:229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA-like format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

[0171] In further embodiments, oligonucleotides or longer fragmentsderived from any of the polynucleotide sequences described herein may beused as targets in a microarray. The microarray can be used to monitorthe expression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

[0172] Microarrays may be prepared, used, and analyzed using methodsknown in the art. (See, e.g., Brennan et al. (1995) U.S. Pat. No.5,474,796; Schena et al. (1996) Proc Natl Acad Sci 93:10614-10619;Baldeschweiler et al. (1995) WO95/251116; Shalon et al. (1995)WO95/35505; Heller et al. (1997) Proc Natl Acad Sci 94:2150-2155; andHeller et al. U.S. Pat. No. 5,605,662.)

[0173] In another embodiment of the invention, nucleic acid sequencesencoding HSLP may be used to generate hybridization probes useful inmapping the naturally occurring genomic sequence. The sequences may bemapped to a particular chromosome, to a specific region of a chromosome,or to artificial chromosome constructions, e.g., HACs, yeast artificialchromosomes (YACs), bacterial artificial chromosomes (BACs), bacterialP1 constructions, or single chromosome cDNA libraries. (See, e.g., Price(1993) Blood Rev 7:127-134, and Trask (1991) Trends Genet 7:149-154.)

[0174] Fluorescent in situ hybridization (FISH) may be correlated withother physical chromosome mapping techniques and genetic map data. (See,e.g., Heinz-Ulrich et al. (1995) In: Meyers, Molecular Biology andBiotechnology, VCH Publishers, New York N.Y., pp. 965-968.) Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding HSLP on a physical chromosomal map and aspecific disorder, or a predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the invention may be used to detect differences in genesequences among normal, carrier, and affected individuals.

[0175] In situ hybridization of chromosomal preparations and physicalmapping techniques, such as linkage analysis using establishedchromosomal markers, may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms by physical mapping. This provides valuableinformation to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, e.g., AT to 11q22-23, any sequences mappingto that area may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti et al. (1988) Nature 336:577-580.) Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,and the like, among normal, is carrier, or affected individuals.

[0176] In another embodiment of the invention, HSLP, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between HSLPand the agent being tested may be measured.

[0177] Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen et al. (1984) WO84/03564.) In thismethod, large numbers of different small test compounds are synthesizedon a solid substrate, such as plastic pins or some other surface. Thetest compounds are reacted with HSLP, or fragments thereof, and washed.Bound HSLP is then detected by methods well known in the art. PurifiedHSLP can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

[0178] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding HSLPspecifically compete with a test compound for binding HSLP. In thismanner, antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with HSLP.

[0179] In additional embodiments, the nucleotide sequences which encodeHSLP may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0180] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0181] I. BRSTNOT24 cDNA Library Construction

[0182] The BRSTNOT24 cDNA library was constructed from diseased breasttissue removed from a 46-year-old Caucasian female during bilateralsubcutaneous mammectomy, bilateral breast augmentation, and total breastreconstruction. Pathology indicated benign fibrocystic diseasebilaterally. The patient presented with fibrosclerosis of the breast.Family history included breast cancer in the mother and sibling.

[0183] The frozen tissue was homogenized and lysed in TRIZOL reagent (1gm tissue/10 ml 1; Life Technologies) using a POLYTRON homogenizer(PT-3000 homogenizer; Brinkmann Instruments, Westbury N.Y.). After briefincubation on ice, chloroform was added (1:5 v/v), and the mixture wascentrifuged to separate the phases. The aqueous phase was removed to afresh tube, and isopropanol was added to precipitate the RNA. The RNAwas resuspended in RNase-free water and treated with DNase. The RNA wasre-extracted once with acid phenol-chloroform and reprecipitated withsodium acetate and ethanol. Poly(A+) RNA was isolated using the OLIGOTEXkit (Qiagen, Chatsworth, Calif.).

[0184] Poly(A+) RNA was used to construct the BRSTNOT24 cDNA libraryaccording to the recommended protocols in the SUPERSCRIPT plasmid system(Life Technologies). The cDNAs were fractionated on a SEPHAROSE CL4Bcolumn (APB), and those cDNAs exceeding 400 bp were ligated into thepINCY plasmid (Incyte Corporation, Palo Alto Calif.). The plasmid wassubsequently transformed into DH5α competent cells (Life Technologies).

[0185] II. Isolation and Sequencing of cDNA Clones

[0186] Plasmid DNA was released from the cells and purified using theR.E.A.L. PREP 96-well plasmid purification kit (Qiagen). The recommendedprotocol was employed except for the following changes: 1) the bacteriawere cultured in 1 ml of sterile Terrific Broth (Life Technologies) withcarbenicillin (Carb) at 25 mg/l and glycerol at 0.4%; 2) after thecultures were incubated for 19 hours, the cells were lysed with 0.3 mlof lysis buffer; and 3) following isopropanol precipitation, the plasmidDNA was resuspended in 0.1 ml of distilled water. The DNA was stored at4° C.

[0187] The cDNAs were prepared using a MICROLAB 2200 system (Hamilton)in combination with DNA ENGINE thermal cyclers (M J Research) andsequenced by the method of Sanger et al. (1975, J Mol Biol 94:441f),using ABI PRISM 377 DNA sequencing systems (PE Biosystems).

[0188] III. Homology Searching of cDNA Clones and Their Deduced Proteins

[0189] The nucleotide sequences and/or amino acid sequences of theSequence Listing were used to query sequences in the GenBank, SwissProt,BLOCKS, and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofhomology using BLAST (Basic Local Alignment Search Tool). (See, e.g.,Altschul (1993) J Mol Evol 36:290-300, and Altschul et al. (1990) J MolBiol 215:403-410.)

[0190] BLAST produced alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST was especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal, or plant) origin. Otheralgorithms could have been used when dealing with primary sequencepatterns and secondary structure gap penalties. (See, e.g., Smith et al.(1992) Protein Engineering 5:35-51.) The sequences disclosed in thisapplication have lengths of at least 49 nucleotides and have no morethan 12% uncalled bases (where N is recorded rather than A, C, G, or T).

[0191] The BLAST approach searched for matches between a query sequenceand a database sequence. BLAST evaluated the statistical significance ofany matches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻⁸ for peptides.

[0192] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and other mammalian sequences(mam). Deduced amino acid sequences from the same clones were thensearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp), for homology.

[0193] Additionally, sequences identified from cDNA libraries may beanalyzed to identify those gene sequences encoding conserved proteinmotifs using an appropriate analysis program, e.g., BLOCK 2 bioanalysisprogram (Incyte Corporation). This motif analysis program, based onsequence information contained in the Swiss-Prot Database and PROSITE,is a method of determining the function of uncharacterized proteinstranslated from genornic or cDNA sequences. (See, e.g., Bairoch et al.(1997) Nucleic Acids Res 25:217-221, and Attwood et al. (1997) J ChemInf Comput Sci 37:417-424.) PROSITE may be used to identify commonfunctional or structural domains in divergent proteins. The method isbased on weight matrices. Motifs identified by this method are thencalibrated against the SWISS-PROT database in order to obtain a measureof the chance distribution of the matches.

[0194] In another alternative, Hidden Markov models (HMMs) may be usedto find protein domains, each defined by a dataset of proteins known tohave a common biological function. (See, e.g., Pearson, and Lipman(1988) Proc Natl Acad Sci 85:2444-2448, and Smith and Waterman (1981) JMol Biol 147:195-197.) HMMs were initially developed to examine speechrecognition patterns, but are now being used in a biological context toanalyze protein and nucleic acid sequences as well as to model proteinstructure. (See, e.g., Krogh et al. (1994) J Mol Biol 235:1501-1531, andCollin et al. (1993) Protein Sci 2:305-314.) HMMs have a formalprobabilistic basis and use position-specific scores for amino acids ornucleotides. The algorithm continues to incorporate information fromnewly identified sequences to increase its motif analysis capabilities.

[0195] IV. Northern Analysis

[0196] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound. (See, e.g., Sambrook,supra, ch. 7; and Ausubel, supra.)

[0197] Analogous computer techniques applying BLAST are used to searchfor identical or related molecules in nucleotide databases such asGenBank or LIFESEQ database (Incyte Corporation). This analysis is muchfaster than multiple membrane-based hybridizations. In addition, thesensitivity of the computer search can be modified to determine whetherany particular match is categorized as exact or homologous.

[0198] The basis of the search is the product score, which is definedas:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0199] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and, with a product score of 70, the match will beexact. Homologous molecules are usually identified by selecting thosewhich show product scores between 15 and 40, although lower scores mayidentify related molecules.

[0200] The results of northern analysis are reported as a list oflibraries in which the transcript encoding HSLP occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0201] V. Extension of HSLP Encoding Polynucleotides

[0202] The nucleic acid sequence of Incyte Clone 3769729 was used todesign oligonucleotide primers for extending a partial nucleotidesequence to full length. One primer was synthesized to initiateextension of an antisense polynucleotide, and the other was synthesizedto initiate extension of a sense polynucleotide. Primers were used tofacilitate the extension of the known sequence “outward” generatingamplicons containing new unknown nucleotide sequence for the region ofinterest. The initial primers were designed from the cDNA using OLIGO4.06 primer analysis software (National Biosciences), or anotherappropriate program, to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to about 72° C. Any stretch ofnucleotides which would result in hairpin structures and primer-primerdimerizations was avoided.

[0203] Selected human cDNA libraries (Life Technologies) were used toextend the sequence. If more than one extension is necessary or desired,additional sets of primers are designed to further extend the knownregion.

[0204] High fidelity amplification was obtained by following theinstructions for the XL-PCR kit (PE Biosystems) and thoroughly mixingthe enzyme and reaction mix. PCR was performed using the DNA ENGINEthermal cycler (M J Research), beginning with 40 pmol of each primer andthe recommended concentrations of all other components of the kit, withthe following parameters: Step 1, 94° C. for 1 min (initialdenaturation); Step 2, 65° C. for 1 min; Step 3, 68° C. for 6 min; Step4, 94° C. for 15 sec; Step 5, 65° C. for 1 min; Step 6, 68° C. for 7min; Step 7, Repeat steps 4 through 6 for an additional 15 cycles; Step8, 94° C. for 15 sec; Step 9, 65° C. for 1 min; Step 10, 68° C. for 7:15min; Step 11, Repeat steps 8 through 10 for an additional 12 cycles;Step 12, 72° C. for 8 min; and Step 13, hold at 4° C.

[0205] A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQUICK DNA purification kit (Qiagen), andtrimmed of overhangs using Klenow enzyme to facilitate religation andcloning.

[0206] After ethanol precipitation, the products were redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium (Sambrook, supra,Appendix A, p. 2). After incubation for one hour at 37° C., the E. colimixture was plated on Luria Bertani (LB) agar (Sambrook, supra, AppendixA, p. 1) containing 2×Carb. The following day, several colonies wererandomly picked from each plate and cultured in 150 μl of liquidLB/2×Carb medium placed in an individual well of an appropriatecommercially-available sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture was transferred into a non-sterile96-well plate and, after dilution 1:10 with water, 5 μl from each samplewas transferred into a PCR array.

[0207] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionwere added to each well. Amplification was performed using the followingconditions: Step 1, 94° C. for 60 sec; Step 2, 94° C. for 20 sec; Step3, 55° C. for 30 sec; Step 4, 72° C. for 90 sec; Step 5, Repeat steps 2through 4 for an additional 29 cycles, Step 6, 72° C. for 180 sec; andStep 7, hold at 4° C.

[0208] Aliquots of the PCR reactions were run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products werecompared to the original partial cDNAs, and appropriate clones wereselected, ligated into plasmid, and sequenced.

[0209] In like manner, the nucleotide sequence of SEQ ID NO:2 is used toobtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for 5′ extension, and an appropriate genomiclibrary.

[0210] VI. Labeling and Use of Individual Hybridization Probes

[0211] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 primer analysis software (National Biosciences) andlabeled by combining 50 pmol of each oligomer, 250 μCi of [γ-³²P]adenosine triphosphate (APB), and T4 polynucleotide kinase (NEN LifeScience Products, Boston Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine resin column(APB). An aliquot containing 10⁷ counts per minute of the labeled probeis used in a typical membrane-based hybridization analysis of humangenomic DNA digested with one of the following endonucleases: Ase I, BglII, Eco RI, Pst I, Xba I, or Pvu II (NEN Life Science Products).

[0212] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to NYTRAN PLUS nylon membranes (Schleicher &Schuell, Durham N. H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARautoradiography film (Eastman Kodak, Rochester N.Y.) is exposed to theblots, hybridization patterns are compared.

[0213] VII. Microarrays

[0214] A chemical coupling procedure and an ink jet device can be usedto synthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

[0215] Full-length cDNAs or fragments thereof may comprise the elementsof the microarray. Fragments suitable for hybridization can be selectedusing software well known in the art such as LASERGENE software(DNASTAR). cDNAs corresponding to one of the nucleotide sequences of thepresent invention, or selected at random from a cDNA library relevant tothe present invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., UVcross-linking followed by thermal and chemical treatments and subsequentdrying. (See, e.g., Schena et al. (1995) Science 270:467-470, and Shalonet al. (1996) Genome Res 6:639-645.) Fluorescent probes are prepared andused for hybridization to the elements on the substrate. The substrateis analyzed by procedures described above.

[0216] VIII. Complementary Polynucleotides

[0217] Sequences complementary to the HSLP-encoding sequences, or anyparts thereof, are used to detect, decrease, or inhibit expression ofnaturally occurring HSLP. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 primeranalysis software and the coding sequence of HSLP. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the HSLP-encoding transcript.

[0218] IX. Expression of HSLP

[0219] Expression of HSLP is accomplished by subcloning the cDNA into anappropriate vector and transforming the vector into host cells. Thisvector contains an appropriate promoter, e.g., β-galactosidase, upstreamof the cloning site, operably associated with the cDNA of interest.(See, e.g., Sambrook, supra, pp. 404-433; and Rosenberg et al (1983)Methods Enzymol 101:123-138.)

[0220] Induction of an isolated, transformed bacterial strain withisopropyl beta-D-thiogalactopy-ranoside using standard methods producesa fusion protein which consists of the first 8 residues ofβ-galactosidase, about 5 to 15 residues of linker, and the protein. Thesignal residues direct the secretion of HSLP into bacterial growth mediawhich can be used directly in the following assay for activity.

[0221] X. Demonstration of HSLP Activity

[0222] An assay for HSLP activity measures its affinity for proteinsinvolved in RNA processing. The yeast two-hybrid system is a sensitive,enzymatic method for detection of protein-protein interactions in vivo.This method is used to identify SMN-interacting proteins (Liu andDreyfuss, supra) and is well known by those skilled in the art.Recombinant DNA methods are used to express HSLP, fibrillarin, and theRGG RNA-binding motif of hnRNP U (RGG) in the yeast Saccharomycescerevisiae. These proteins are expressed as fusions with other proteinfragments involved in gene regulation. The interaction of HSLP witheither fibrillarin or RGG triggers the expression of a reporter gene.This gene encodes a metabolic enzyme that generates a colored reactionproduct. When plated on the appropriate substratum, the yeast will turnfrom white to blue in color. The amount of reaction product can bequantified spectrophotometrically and is proportional to the affinity ofHSLP for either fibrillarin or RGG.

[0223] XI. Production of HSLP Specific Antibodies

[0224] HSLP substantially purified using PAGE electrophoresis(Harrington (1990) Methods Enzymol 182:488-495), or other purificationtechniques, is used to immunize rabbits and to produce antibodies usingstandard protocols well known in the art. The HSLP amino acid sequenceis analyzed using LASERGENE software (DNASTAR) to determine regions ofhigh immunogenicity, and a corresponding oligopeptide is synthesized andused to raise antibodies. Methods for selection of appropriate epitopes,such as those near the C-terminus or in hydrophilic regions are welldescribed in Ausubel (supra).

[0225] Typically, the oligopeptides are 15 residues in length, and aresynthesized using an ABI 431A peptide synthesizer using Fmoc-chemistryand coupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester to increaseimmunogenicity. (See Ausubel, supra.) Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio-iodinated goat anti-rabbitIgG.

[0226] XII. Purification of Naturally Occurring HSLP Using SpecificAntibodies

[0227] Naturally occurring or recombinant HSLP is substantially purifiedby immunoaffinity chromatography using antibodies specific for HSLP. Animmunoaffinity column is constructed by covalently coupling anti-HSLPantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (APB). After the coupling, the resin is blocked and washedaccording to the manufacturer's instructions.

[0228] Media containing HSLP are passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of HSLP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/HSLP binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and HSLPis collected.

[0229] XIII. Identification of Molecules Which Interact with HSLP

[0230] HSLP, or biologically active fragments thereof, are labeled with1211 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem J133:529-539.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled HSLP, washed, and anywells with labeled HSLP complex are assayed. Data obtained usingdifferent concentrations of HSLP are used to calculate values for thenumber, affinity, and association of HSLP with the candidate molecules

[0231] Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1 4 238 amino acids amino acid single linear BRSTNOT24 3769729 1 Met SerGlu Asp Leu Ala Lys Gln Leu Ala Ser Tyr Lys Ala Gln Leu 1 5 10 15 GlnGln Val Glu Ala Ala Leu Ser Gly Asn Gly Glu Asn Glu Asp Leu 20 25 30 LeuLys Leu Lys Lys Asp Leu Gln Glu Val Ile Glu Leu Thr Lys Asp 35 40 45 LeuLeu Ser Thr Gln Pro Ser Glu Thr Leu Ala Ser Ser Asp Ser Phe 50 55 60 AlaSer Thr Gln Pro Thr His Ser Trp Lys Val Gly Asp Lys Cys Met 65 70 75 80Ala Val Trp Ser Glu Asp Gly Gln Cys Tyr Glu Ala Glu Ile Glu Glu 85 90 95Ile Asp Glu Glu Asn Gly Thr Ala Ala Ile Thr Phe Ala Gly Tyr Gly 100 105110 Asn Ala Glu Val Thr Pro Leu Leu Asn Leu Lys Pro Val Glu Glu Gly 115120 125 Arg Lys Ala Lys Glu Asp Ser Gly Asn Lys Pro Met Ser Lys Lys Glu130 135 140 Met Ile Ala Gln Gln Arg Glu Tyr Lys Lys Lys Lys Ala Leu LysLys 145 150 155 160 Ala Gln Arg Ile Lys Glu Leu Glu Gln Glu Arg Glu AspGln Lys Val 165 170 175 Lys Trp Gln Gln Phe Asn Asn Arg Ala Tyr Ser LysAsn Lys Lys Gly 180 185 190 Gln Val Lys Arg Ser Ile Phe Ala Ser Pro GluSer Val Thr Gly Lys 195 200 205 Val Gly Val Gly Thr Cys Gly Ile Ala AspLys Pro Met Thr Gln Tyr 210 215 220 Gln Asp Thr Ser Lys Tyr Asn Val ArgHis Leu Met Pro Gln 225 230 235 2426 base pairs nucleic acid singlelinear BRSTNOT24 3769729 2 TCTTTCATAG AGACTAAAGT TATTCAGCAG GCAGCAAAATAATCTACTTA AGTCCTGCCT 60 TTCTTTTTTC ACTTAAAAAA GTGGGTGTGA TAATATCCAGGCTAGCTAGC TGACTAGCTC 120 CCCGGGCAGT CTATGATAAT CAGAGATAGT CAATTTATTAGGCTGTTTTG CTGAATAAGC 180 TGGTTCTAAA GGAGGCAGGG GTCAAGTCAC TTGTCTCATATATTACAGTG GCTCTCTGCA 240 TCCCCGAAAC GCCTTCCTTC AGTAAGCAGA GTGCTTGAGTGCACCCCATT TGACCTGCTG 300 ATATGTAGAT CACAACNCCT GATGCTTCCT GGAATTGCCGATTACTGTAA CTGCTGCCCA 360 TCTGTCGATG AAGGAGCAGT TTCAGAACTC AGACTTGAGGGAGGAAAAGT AATTAATGGT 420 GCCCGGCGTT CCTCCCTTCC CCCTCGCCGC CGACCGAGTTCTTCCTTTTC AGACCGGGTC 480 GCCTTGCTGT CGTCGCGGTG ATTTTCCTGC TACTGCTACTGCTGCTGCTG CCACCGCCAC 540 TACCACTGGG CTCATTTGCC CCGACCCCTT CCCGCCGCCCCGCCCCCAGC CCCACACAAG 600 ATGTCAGAGG ATTTAGCAAA GCAGCTGGCA AGCTACAAAGCTCAGCTCCA GCAAGTTGAA 660 GCTGCATTAT CTGGAAATGG AGAAAATGAA GATTTGCTAAAATTGAAGAA AGATTTACAA 720 GAAGTTATAG AACTAACCAA AGACCTTCTG TCAACTCAACCTTCTGAGAC GCTTGCAAGT 780 TCAGACAGTT TTGCTTCTAC TCAACCTACT CATTCATGGAAAGTAGGAGA CAAGTGTATG 840 GCAGTCTGGA GTGAAGATGG ACAGTGTTAT GAAGCGGAGATTGAGGAGAT AGATGAAGAA 900 AATGGCACCG CTGCAATCAC CTTTGCTGGT TATGGCAATGCTGAAGTGAC TCCACTGTTG 960 AACCTCAAGC CTGTAGAAGA AGGAAGGAAG GCAAAGGAGGACAGTGGCAA CAAACCCATG 1020 TCAAAAAAAG AAATGATTGC CCAGCAGCGT GAATATAAAAAGAAGAAAGC TTTGAAAAAA 1080 GCTCAGAGAA TAAAAGAACT TGAGCAGGAA AGAGAGGACCAGAAAGTGAA ATGGCAACAA 1140 TTCAACAACA GAGCCTATTC TAAAAACAAA AAAGGCCAGGTAAAGAGGAG TATTTTTGCT 1200 TCACCTGAGA GTGTGACTGG TAAAGTTGGA GTAGGAACCTGTGGAATTGC TGATAAACCT 1260 ATGACACAAT ATCAAGATAC CTCTAAATAC AATGTCAGGCATTTGATGCC TCAATAATCA 1320 GAAAAACTGT TGGATTTCAT CTCTGCAGGG CTTTACATTTACCTTTTTAT CCTTATATTT 1380 TTCTAAAGGT AAATTATTTG TTAGATGAGT AAGCAAGATACCATTGTCGT CATTGGTTGG 1440 CTTCAGTAGA ATGAAACGTG AAGAAATTGC ATTTGATAACTGCTATTCAT TTAACTTTTC 1500 TCATTATCAG TACCACGGTT CCCTCAAAGT TTGTTGAATAAAGCAACTTT TGTAGATGCT 1560 GTTTCATACA GCACTTAGAT GAATTATTGA TCTTCCTAATATCAGGCGCC TACTTAACCT 1620 ATGGTGTGTA CTTTTTGTAA GTTGTAACTT GAAATTTTCAGATGCTTTGA ACTTGACACA 1680 TACTCTAGCA ATTCATTGGA ACACCAAGGC AAAAACACCAACCTGCTAAA AGAGATCTTT 1740 TCATTTTTCT TATTTTCAGC TTTAAAACTT AGCTGTCGTTCAGTTAAGCT TAAAGATAGG 1800 TTAATTTGTA AATGGCAAAG TTTGTTTTGA GGTTTTTCCTCAATAACTTG TTTCCCAGGC 1860 CTATTAGGCC ATCTCTAAAA TTGATCTAGC TGTTTTATTTTTATGTACTC TTAGTTTTAT 1920 GTAAGAAACC TTAGGATGAG CTCCCTTTTC TAAGGTGTTTTTGTTTTTTG TATGTTTGCT 1980 TTTTTCCTGT TTTTTGTTTT TTCCATTTAC GGCAGTGGTACCATGTTTTG GATGTGTGAT 2040 GTTTATATGG GAGAACAAAA AGCTGATGTA TAGCCCTGTATACAGTGTAG ATACTATTTT 2100 TGTAAAAACA CAAGGCTAAA TTAATGAACA AGAATACTGAATATTTCATC ATTAAAAATT 2160 TCTTGTATTT CTTGTGCATT AATCTGACGA TAATTTCCCTGTATATTATG TTCATTTAGC 2220 TGTTTGTAAT TTTTGTTAAT TAGATCAGGT TGTCTGCATTTGTTGGTGTA AGTGAACATC 2280 ATCACAGTTA TCCTGAGTTG AGTTTAAGCC AAATACATGCATAGAAAAGG GTCTTCCTAT 2340 TAATGGAAGA AGGTAATTTT TAGGATGTGT ATTATTTCAGTTTTGTATGT TTAACTTTTA 2400 TTAAATAAAG TGTTTTTAAA ATCTCC 2426 288 aminoacids amino acid single linear GenBank 1857114 3 Met Ala Met Gly Ser GlyGly Ala Gly Ser Glu Gln Glu Asp Thr Val 1 5 10 15 Leu Phe Arg Arg GlyThr Gly Gln Ser Asp Asp Ser Asp Ile Trp Asp 20 25 30 Asp Thr Ala Leu IleLys Ala Tyr Asp Lys Ala Val Ala Ser Phe Lys 35 40 45 His Ala Leu Lys AsnGly Asp Ile Cys Glu Thr Pro Asp Lys Pro Lys 50 55 60 Gly Thr Ala Arg ArgLys Pro Ala Lys Lys Asn Lys Ser Gln Lys Lys 65 70 75 80 Asn Ala Thr ThrPro Leu Lys Gln Trp Lys Val Gly Asp Lys Cys Ser 85 90 95 Ala Val Trp SerGlu Asp Gly Cys Ile Tyr Pro Ala Thr Ile Thr Ser 100 105 110 Ile Asp PheLys Arg Glu Thr Cys Val Val Val Tyr Thr Gly Tyr Gly 115 120 125 Asn ArgGlu Glu Gln Asn Leu Ser Asp Leu Leu Ser Pro Thr Cys Glu 130 135 140 ValAla Asn Ser Thr Glu Gln Asn Thr Gln Glu Asn Glu Ser Gln Val 145 150 155160 Ser Thr Asp Asp Ser Glu His Ser Ser Arg Ser Leu Arg Ser Lys Ala 165170 175 His Ser Lys Ser Lys Ala Ala Pro Trp Thr Ser Phe Leu Pro Pro Pro180 185 190 Pro Pro Met Pro Gly Ser Gly Leu Gly Pro Gly Lys Pro Gly LeuLys 195 200 205 Phe Asn Gly Pro Pro Pro Pro Pro Pro Leu Pro Pro Pro ProPhe Leu 210 215 220 Pro Cys Trp Met Pro Pro Phe Pro Ser Gly Pro Pro IleIle Pro Pro 225 230 235 240 Pro Pro Pro Ile Ser Pro Asp Cys Leu Asp AspThr Asp Ala Leu Gly 245 250 255 Ser Met Leu Ile Ser Trp Tyr Met Ser GlyTyr His Thr Gly Tyr Tyr 260 265 270 Met Gly Phe Arg Gln Asn Lys Lys GluGly Lys Cys Ser His Thr Asn 275 280 285 294 amino acids amino acidsingle linear GenBank 1314346 4 Met Ala Met Ser Ser Gly Gly Ser Gly GlyGly Val Pro Glu Gln Glu 1 5 10 15 Asp Ser Val Leu Phe Arg Arg Gly ThrGly Gln Ser Asp Asp Ser Asp 20 25 30 Ile Trp Asp Asp Thr Ala Leu Ile LysAla Tyr Asp Lys Ala Val Ala 35 40 45 Ser Phe Lys His Ala Leu Lys Asn GlyAsp Ile Cys Glu Thr Ser Gly 50 55 60 Lys Pro Lys Thr Thr Pro Lys Arg LysPro Ala Lys Lys Asn Lys Ser 65 70 75 80 Gln Lys Lys Asn Thr Ala Ala SerLeu Gln Gln Trp Lys Val Gly Asp 85 90 95 Lys Cys Ser Ala Ile Trp Ser GluAsp Gly Cys Ile Tyr Pro Ala Thr 100 105 110 Ile Ala Ser Ile Asp Phe LysArg Glu Thr Cys Val Val Val Tyr Thr 115 120 125 Gly Tyr Gly Asn Arg GluGlu Gln Asn Leu Ser Asp Leu Leu Ser Pro 130 135 140 Ile Cys Glu Val AlaAsn Asn Ile Glu Gln Asn Ala Gln Glu Asn Glu 145 150 155 160 Asn Glu SerGln Val Ser Thr Asp Glu Ser Glu Asn Ser Arg Ser Pro 165 170 175 Gly AsnLys Ser Asp Asn Ile Lys Pro Lys Ser Ala Pro Trp Asn Ser 180 185 190 PheLeu Pro Pro Pro Pro Pro Met Pro Gly Pro Arg Leu Gly Pro Gly 195 200 205Lys Pro Gly Leu Lys Phe Asn Gly Pro Pro Pro Pro Pro Pro Pro Pro 210 215220 Pro Pro His Leu Leu Ser Cys Trp Leu Pro Pro Phe Pro Ser Gly Pro 225230 235 240 Pro Ile Ile Pro Pro Pro Pro Pro Ile Cys Pro Asp Ser Leu AspAsp 245 250 255 Ala Asp Ala Leu Gly Ser Met Leu Ile Ser Trp Tyr Met SerGly Tyr 260 265 270 His Thr Gly Tyr Tyr Met Gly Phe Arg Gln Asn Gln LysGlu Gly Arg 275 280 285 Cys Ser His Ser Leu Asn 290

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence of SEQID NO:1, b) a polypeptide comprising a naturally occurring an amino acidsequence at least 90% identical to the amino acid sequence of SEQ IDNO:1, c) a biologically active fragment of a polypeptide having an aminoacid sequence of SEQ ID NO:1, and d) an immunogenic fragment of apolypeptide having an amino acid sequence of SEQ ID NO:1.
 2. An isolatedpolypeptide of claim 1, comprising the amino acid sequence of SEQ IDNO:1.
 3. An isolated polynucleotide encoding a polypeptide of claim 1.4. An isolated polynucleotide encoding a polypeptide of claim
 2. 5. Anisolated polynucleotide of claim 4 comprising the polynucleotidesequence of SEQ ID NO:2.
 6. A recombinant polynucleotide comprising apromoter sequence operably linked to a polynucleotide of claim
 3. 7. Acell transformed with a recombinant polynucleotide of claim
 6. 8. Atransgenic organism comprising a recombinant polynucleotide of claim 6.9. A method of producing a polypeptide of claim 1, the methodcomprising: a) culturing a cell under conditions suitable for expressionof the polypeptide, wherein said cell is transformed with a recombinantpolynucleotide, and said recombinant polynucleotide comprises a promotersequence operably linked to a polynucleotide encoding the polypeptide ofclaim 1, and b) recovering the polypeptide so expressed.
 10. A method ofclaim 9, wherein the polypeptide comprises the amino acid sequence ofSEQ ID NO:1.
 11. An isolated antibody which specifically binds to apolypeptide of claim
 1. 12. An isolated polynucleotide selected from thegroup consisting of: a) a polynucleotide comprising a polynucleotidesequence of SEQ ID NO:2, b) a polynucleotide comprising a naturallyoccurring polynucleotide sequence at least 90% identical to thepolynucleotide sequence of SEQ ID NO:2, c) a polynucleotidecomplementary to a polynucleotide of a), d) a polynucleotidecomplementary to a polynucleotide of b) and e) an RNA equivalent ofa)-d).
 13. An isolated polynucleotide comprising at least 60 contiguousnucleotides of a polynucleotide of claim
 12. 14. A method of detecting atarget polynucleotide in a sample, said target polynucleotide having asequence of a polynucleotide of claim 12, the method comprising: a)hybridizing the sample with a probe comprising at least 20 contiguousnucleotides comprising a sequence complementary to said targetpolynucleotide in the sample, and which probe specifically hybridizes tosaid target polynucleotide, under conditions whereby a hybridizationcomplex is formed between said probe and said target polynucleotide orfragments thereof, and b) detecting the presence or absence of saidhybridization complex, and, optionally, if present, the amount thereof.15. A method of claim 14, wherein the probe comprises at least 60contiguous nucleotides.
 16. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide comprises the aminoacid sequence of SEQ ID NO:1.
 19. A method for treating a disease orcondition associated with decreased expression of functional HSLP,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method of screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) contacting a sample comprising a polypeptide of claim 1with a compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional HSLP, comprising administering to a patient in need of suchtreatment a composition of claim
 21. 23. A method of screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) contacting a sample comprising a polypeptideof claim 1 with a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional HSLP, comprising administering to a patientin need of such treatment a composition of claim
 24. 26. A method ofscreening for a compound that specifically binds to the polypeptide ofclaim 1, the method comprising: a) combining the polypeptide of claim 1with at least one test compound under suitable conditions, and b)detecting binding of the polypeptide of claim 1 to the test compound,thereby identifying a compound that specifically binds to thepolypeptide of claim
 1. 27. A method of screening for a compound thatmodulates the activity of the polypeptide of claim 1, said methodcomprising: a) combining the polypeptide of claim 1 with at least onetest compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method of screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a polynucleotide sequence of claim 5, themethod comprising: a) contacting a sample comprising the targetpolynucleotide with, under conditions suitable for the expression of thetarget polynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 29. A method of screening for potentialtoxicity of a test compound, the method comprising: a) treating abiological sample containing nucleic acids with the test compound, b)hybridizing the nucleic acids of the treated biological sample with aprobe comprising at least 20 contiguous nucleotides of a polynucleotideof claim 12 under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide comprising a polynucleotide sequenceof a polynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample indicates potential toxicity of the test compound. 30.A diagnostic test for a condition or disease associated with theexpression of HSLP in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of HSLP in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, further comprising a label.
 35. A method ofdiagnosing a condition or disease associated with the expression of HSLPin a subject, comprising administering to said subject an effectiveamount of the composition of claim
 34. 36. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 11,the method comprising: a) immunizing an animal with a polypeptideconsisting of the amino acid sequence of SEQ ID NO:1, or an immunogenicfragment thereof, under conditions to elicit an antibody response, b)isolating antibodies from said animal, and c) screening the isolatedantibodies with the polypeptide, thereby identifying a polyclonalantibody which binds specifically to a polypeptide comprising the aminoacid sequence of SEQ ID NO:1.
 37. A polyclonal antibody produced by amethod of claim
 36. 38. A composition comprising the polyclonal antibodyof claim 37 and a suitable carrier.
 39. A method of making a monoclonalantibody with the specificity of the antibody of claim 11, the methodcomprising: a) immunizing an animal with a polypeptide consisting of theamino acid sequence of SEQ ID NO:1, or an immunogenic fragment thereof,under conditions to elicit an antibody response, b) isolating antibodyproducing cells from the animal, c) fusing the antibody producing cellswith immortalized cells to form monoclonal antibody-producing hybridomacells, d) culturing the hybridoma cells, and e) isolating from theculture monoclonal antibody which binds specifically to a polypeptidecomprising the amino acid sequence of SEQ ID NO:1.
 40. A monoclonalantibody produced by a method of claim
 39. 41. A composition comprisingthe monoclonal antibody of claim 40 and a suitable carrier.
 42. Theantibody of claim 11, wherein the monoclonal antibody is produced byscreening a Fab expression library.
 43. The antibody of claim 11,wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 44. A method of detecting a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 in a sample, themethod comprising: a) incubating the antibody of claim 11 with a sampleunder conditions to allow specific binding of the antibody and thepolypeptide, and b) detecting specific binding, wherein specific bindingindicates the presence of a polypeptide comprising the amino acidsequence of SEQ ID NO:1 in the sample.
 45. A method of purifying apolypeptide comprising an amino acid sequence of SEQ ID NO:1 from asample, the method comprising: a) incubating the antibody of claim 11with a sample under conditions to allow specific binding of the antibodyand the polypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide comprising the amino acid sequence ofSEQ ID NO:1.
 46. A microarray wherein at least one element of themicroarray is a polynucleotide of claim
 13. 47. A method of generatingan expression profile of a sample which contains polynucleotides, themethod comprising: a) labeling the polynucleotides of the sample, b)contacting the elements of the microarray of claim 46 with the labeledpolynucleotides of the sample under conditions suitable for theformation of a hybridization complex, and c) quantifying the expressionof the polynucleotides in the sample.
 48. An array comprising differentnucleotide molecules affixed in distinct physical locations on a solidsubstrate, wherein at least one of said nucleotide molecules comprises afirst oligonucleotide or polynucleotide sequence specificallyhybridizable with at least 30 contiguous nucleotides of a targetpolynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:1.
 57. A polynucleotide of claim 12, comprisingthe polynucleotide sequence of SEQ ID NO:2.